U.S. patent application number 12/057642 was filed with the patent office on 2008-07-24 for leucine-based motif and clostridial neurotoxins.
This patent application is currently assigned to Allergan, Inc.. Invention is credited to Kei Roger Aoki, Todd M. Herrington, Lance E. Steward.
Application Number | 20080177042 12/057642 |
Document ID | / |
Family ID | 24487624 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080177042 |
Kind Code |
A1 |
Steward; Lance E. ; et
al. |
July 24, 2008 |
LEUCINE-BASED MOTIF AND CLOSTRIDIAL NEUROTOXINS
Abstract
Modified neurotoxin comprising neurotoxin including structural
modification, wherein the structural modification alters the
biological persistence, preferably the biological half-life, of the
modified neurotoxin relative to an identical neurotoxin without the
structural modification. The structural modification includes
addition or deletion of a leucine-based motif or parts thereof. In
one embodiment, methods of making the modified neurotoxin include
using recombinant techniques. In another embodiment, methods of
using the modified neurotoxin to treat biological disorders include
treating autonomic disorders, neuromuscular disorders or pains.
Inventors: |
Steward; Lance E.; (Irvine,
CA) ; Herrington; Todd M.; (Irvine, CA) ;
Aoki; Kei Roger; (Coto de Caza, CA) |
Correspondence
Address: |
ALLERGAN, INC.
2525 DUPONT DRIVE, T2-7H
IRVINE
CA
92612-1599
US
|
Assignee: |
Allergan, Inc.
|
Family ID: |
24487624 |
Appl. No.: |
12/057642 |
Filed: |
March 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11039268 |
Jan 19, 2005 |
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12057642 |
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09620840 |
Jul 21, 2000 |
6903187 |
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11039268 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
Y10S 530/825 20130101;
A61P 25/00 20180101; A61P 5/00 20180101; A61P 25/04 20180101; A61P
29/02 20180101; A61K 38/00 20130101; A61P 21/00 20180101; A61P
27/00 20180101; A61P 27/02 20180101; A61P 37/00 20180101; C07K
14/33 20130101; A61P 1/00 20180101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 14/33 20060101
C07K014/33 |
Claims
1. A modified botulinum neurotoxin type E having increased
biological half-life, wherein the modification comprises the
addition of the leucine-based motif of SEQ ID NO: 1, and wherein
the added leucine-based motif increases biological half-life of the
modified botulinum toxin type E relative to an identical botulinum
toxin type E without the added leucine-based motif.
2. The modified botulinum neurotoxin type E of claim 1, wherein the
added leucine-based motif is SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 16, or SEQ ID NO: 18.
Description
[0001] This application is a divisional and claims priority
pursuant to 35 U.S.C. .sctn. 120 to U.S. patent application Ser.
No. 11/039,268, filed Jan. 19, 2005, a divisional application that
claims priority pursuant to 35 U.S.C. .sctn. 120 to U.S. patent
application Ser. No. 09/620,840, filed Jul. 21, 2000, each of which
is hereby incorporated by reference in its entirety.
BACKGROUND
[0002] The present invention relates to modified neurotoxins,
particularly modified Clostridial neurotoxins, and use thereof to
treat various disorders, including neuromuscular disorders,
autonomic nervous system disorders and pain.
[0003] The clinical use of botulinum toxin serotype A (herein after
"BoNT/A"), a serotype of Clostridial neurotoxin, represents one of
the most dramatic role reversals in modern medicine: a potent
biologic toxin transformed into a therapeutic agent. BoNT/A has
become a versatile tool in the treatment of a wide variety of
disorders and conditions characterized by muscle hyperactivity,
autonomic nervous system hyperactivity and/or pain.
Botulinum toxin
[0004] The anaerobic, gram positive bacterium Clostridium botulinum
produces a potent polypeptide neurotoxin, botulinum toxin, which
causes a neuroparalytic illness in humans and animals referred to
as botulism. The spores of Clostridium botulinum are found in soil
and can grow in improperly sterilized and sealed food containers of
home based canneries, which are the cause of many of the cases of
botulism. The effects of botulism typically appear 18 to 36 hours
after eating the foodstuffs infected with a Clostridium botulinum
culture or spores. The botulinum toxin can apparently pass
unattenuated through the lining of the gut and attack peripheral
motor neurons. Symptoms of botulinum toxin intoxication can
progress from difficulty walking, swallowing, and speaking to
paralysis of the respiratory muscles and death.
[0005] BoNT/A is the most lethal natural biological agent known to
man. About 50 picograms of botulinum toxin (purified neurotoxin
complex) serotype A is a LD.sub.50 in mice. One unit (U) of
botulinum toxin is defined as the LD.sub.50 upon intraperitoneal
injection into female Swiss Webster mice weighing 18-20 grams each.
Seven immunologically distinct botulinum neurotoxins have been
characterized, these being respectively botulinum neurotoxin
serotypes A, B, C.sub.1, D, E, F and G each of which is
distinguished by neutralization with serotype-specific antibodies.
The different serotypes of botulinum toxin vary in the animal
species that they affect and in the severity and duration of the
paralysis they evoke. For example, it has been determined that
BoNt/A is 500 times more potent, as measured by the rate of
paralysis produced in the rat, than is botulinum toxin serotype B
(BoNT/B). Additionally, BoNt/B has been determined to be non-toxic
in primates at a dose of 480 U/kg which is about 12 times the
primate LD.sub.50 for BoNt/A. Botulinum toxin apparently binds with
high affinity to cholinergic motor neurons, is translocated into
the neuron and blocks the release of acetylcholine.
[0006] Botulinum toxins have been used in clinical settings for the
treatment of neuromuscular disorders characterized by hyperactive
skeletal muscles. BoNt/A has been approved by the U.S. Food and
Drug Administration for the treatment of blepharospasm, strabismus
and hemifacial spasm. Non-serotype A botulinum toxin serotypes
apparently have a lower potency and/or a shorter duration of
activity as compared to BoNt/A. Clinical effects of peripheral
intramuscular BoNt/A are usually seen within one week of injection.
The typical duration of symptomatic relief from a single
intramuscular injection of BoNt/A averages about three months.
[0007] Although all the botulinum toxins serotypes apparently
inhibit release of the neurotransmitter acetylcholine at the
neuromuscular junction, they do so by affecting different
neurosecretory proteins and/or cleaving these proteins at different
sites. For example, botulinum serotypes A and E both cleave the 25
kiloDalton (kD) synaptosomal associated protein (SNAP-25), but they
target different amino acid sequences within this protein. BoNT/B,
D, F and G act on vesicle-associated protein (VAMP, also called
synaptobrevin), with each serotype cleaving the protein at a
different site. Finally, botulinum toxin serotype C.sub.1
(BoNT/C.sub.1) has been shown to cleave both syntaxin and SNAP-25.
These differences in mechanism of action may affect the relative
potency and/or duration of action of the various botulinum toxin
serotypes.
[0008] Regardless of serotype, the molecular mechanism of toxin
intoxication appears to be similar and to involve at least three
steps or stages. In the first step of the process, the toxin binds
to the presynaptic membrane of the target neuron through a specific
interaction between the H chain and a cell surface receptor; the
receptor is thought to be different for each serotype of botulinum
toxin and for tetanus toxin. The carboxyl end segment of the H
chain, H.sub.c, appears to be important for targeting of the toxin
to the cell surface.
[0009] In the second step, the toxin crosses the plasma membrane of
the poisoned cell. The toxin is first engulfed by the cell through
receptor-mediated endocytosis, and an endosome containing the toxin
is formed. The toxin then escapes the endosome into the cytoplasm
of the cell. This last step is thought to be mediated by the amino
end segment of the H chain, H.sub.N, which triggers a
conformational change of the toxin in response to a pH of about 5.5
or lower. Endosomes are known to possess a proton pump which
decreases intra endosomal pH. The conformational shift exposes
hydrophobic residues in the toxin, which permits the toxin to embed
itself in the endosomal membrane. The toxin then translocates
through the endosomal membrane into the cytosol.
[0010] The last step of the mechanism of botulinum toxin activity
appears to involve reduction of the disulfide bond joining the H
and L chain. The entire toxic activity of botulinum and tetanus
toxins is contained in the L chain of the holotoxin; the L chain is
a zinc (Zn++) endopeptidase which selectively cleaves proteins
essential for recognition and docking of
neurotransmitter-containing vesicles with the cytoplasmic surface
of the plasma membrane, and fusion of the vesicles with the plasma
membrane. Tetanus neurotoxin, botulinum toxin/B/D,/F, and/G cause
degradation of synaptobrevin (also called vesicle-associated
membrane protein (VAMP)), a synaptosomal membrane protein. Most of
the VAMP present at the cytosolic surface of the synaptic vesicle
is removed as a result of any one of these cleavage events. Each
toxin specifically cleaves a different bond.
[0011] The molecular weight of the botulinum toxin protein
molecule, for all seven of the known botulinum toxin serotypes, is
about 150 kD. Interestingly, the botulinum toxins are released by
Clostridial bacterium as complexes comprising the 150 kD botulinum
toxin protein molecule along with associated non-toxin proteins.
Thus, the BoNt/A complex can be produced by Clostridial bacterium
as 900 kD, 500 kD and 300 kD forms. BoNT/B and C.sub.1 are
apparently produced as only a 500 kD complex. BoNT/D is produced as
both 300 kD and 500 kD complexes. Finally, BoNT/E and F are
produced as only approximately 300 kD complexes. The complexes
(i.e. molecular weight greater than about 150 kD) are believed to
contain a non-toxin hemaglutinin protein and a non-toxin and
non-toxic nonhemaglutinin protein. These two non-toxin proteins
(which along with the botulinum toxin molecule comprise the
relevant neurotoxin complex) may act to provide stability against
denaturation to the botulinum toxin molecule and protection against
digestive acids when toxin is ingested. Additionally, it is
possible that the larger (greater than about 150 kD molecular
weight) botulinum toxin complexes may result in a slower rate of
diffusion of the botulinum toxin away from a site of intramuscular
injection of a botulinum toxin complex.
[0012] In vitro studies have indicated that botulinum toxin
inhibits potassium cation induced release of both acetylcholine and
norepinephrine from primary cell cultures of brainstem tissue.
Additionally, it has been reported that botulinum toxin inhibits
the evoked release of both glycine and glutamate in primary
cultures of spinal cord neurons and that in brain synaptosome
preparations botulinum toxin inhibits the release of each of the
neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and
glutamate.
[0013] BoNt/A can be obtained by establishing and growing cultures
of Clostridium botulinum in a fermenter and then harvesting and
purifying the fermented mixture in accordance with known
procedures. All the botulinum toxin serotypes are initially
synthesized as inactive single chain proteins which must be cleaved
or nicked by proteases to become neuroactive. The bacterial strains
that make botulinum toxin serotypes A and G possess endogenous
proteases and serotypes A and G can therefore be recovered from
bacterial cultures in predominantly their active form. In contrast,
botulinum toxin serotypes C.sub.1, D and E are synthesized by
nonproteolytic strains and are therefore typically unactivated when
recovered from culture. Serotypes B and F are produced by both
proteolytic and nonproteolytic strains and therefore can be
recovered in either the active or inactive form. However, even the
proteolytic strains that produce, for example, the BoNt/B serotype
only cleave a portion of the toxin produced. The exact proportion
of nicked to unnicked molecules depends on the length of incubation
and the temperature of the culture. Therefore, a certain percentage
of any preparation of, for example, the BoNt/B toxin is likely to
be inactive, possibly accounting for the known significantly lower
potency of BoNt/B as compared to BoNt/A. The presence of inactive
botulinum toxin molecules in a clinical preparation will contribute
to the overall protein load of the preparation, which has been
linked to increased antigenicity, without contributing to its
clinical efficacy. Additionally, it is known that BoNt/B has, upon
intramuscular injection, a shorter duration of activity and is also
less potent than BoNt/A at the same dose level. It has been
reported that BoNt/A has been used in clinical settings as
follows:
[0014] (1) about 75-125 units of BOTOX.RTM..sup.1 per intramuscular
injection (multiple muscles) to treat cervical dystonia;
.sup.1Available from Allergan, Inc., of Irvine, Calif. under the
tradename BOTOX.RTM..
[0015] (2) 5-10 units of BOTOX.RTM. per intramuscular injection to
treat glabellar lines (brow furrows) (5 units injected
intramuscularly into the procerus muscle and 10 units injected
intramuscularly into each corrugator supercilii muscle);
[0016] (3) about 30-80 units of BOTOX.RTM. to treat constipation by
intrasphincter injection of the puborectalis muscle;
[0017] (4) about 1-5 units per muscle of intramuscularly injected
BOTOX.RTM. to treat blepharospasm by injecting the lateral
pre-tarsal orbicularis oculi muscle of the upper lid and the
lateral pre-tarsal orbicularis oculi of the lower lid.
[0018] (5) to treat strabismus, extraocular muscles have been
injected intramuscularly with between about 1-5 units of
BOTOX.RTM., the amount injected varying based upon both the size of
the muscle to be injected and the extent of muscle paralysis
desired (i.e. amount of diopter correction desired).
[0019] (6) to treat upper limb spasticity following stroke by
intramuscular injections of BOTOX.RTM. into five different upper
limb flexor muscles, as follows: [0020] (a) flexor digitorum
profundus: 7.5 U to 30 U [0021] (b) flexor digitorum sublimus: 7.5
U to 30 U [0022] (c) flexor carpi ulnaris: 10 U to 40 U [0023] (d)
flexor carpi radialis: 15 U to 60 U [0024] (e) biceps brachii: 50 U
to 200 U. Each of the five indicated muscles has been injected at
the same treatment session, so that the patient receives from 90 U
to 360 U of upper limb flexor muscle BOTOX.RTM. by intramuscular
injection at each treatment session.
[0025] The success of BoNt/A to treat a variety of clinical
conditions has led to interest in other botulinum toxin serotypes.
A study of two commercially available BoNT/A preparations
(BOTOX.RTM. and Dysport.RTM.) and preparations of BoNT/B and F
(both obtained from Wako Chemicals, Japan) has been carried out to
determine local muscle weakening efficacy, safety and antigenic
potential. Botulinum toxin preparations were injected into the head
of the right gastrocnemius muscle (0.5 to 200.0 units/kg) and
muscle weakness was assessed using the mouse digit abduction
scoring assay (DAS). ED.sub.50 values were calculated from dose
response curves. Additional mice were given intramuscular
injections to determine LD.sub.50 doses. The therapeutic index was
calculated as LD.sub.50/ED.sub.50. Separate groups of mice received
hind limb injections of BOTOX.RTM. (5.0 to 10.0 units/kg) or BoNt/B
(50.0 to 400.0 units/kg), and were tested for muscle weakness and
increased water consumption, the later being a putative model for
dry mouth. Antigenic potential was assessed by monthly
intramuscular injections in rabbits (1.5 or 6.5 ng/kg for BoNt/B or
0.15 ng/kg for BOTOX.RTM.). Peak muscle weakness and duration were
dose related for all serotypes. DAS ED.sub.50 values (units/kg)
were as follows: BOTOX.RTM.: 6.7, Dysport.RTM.: 24.7, BoNt/B: 27.0
to 244.0, BoNT/F: 4.3. BOTOX.RTM. had a longer duration of action
than BoNt/B or BoNt/F. Therapeutic index values were as follows:
BOTOX.RTM.: 10.5, Dysport.RTM.: 6.3, BoNt/B: 3.2. Water consumption
was greater in mice injected with BoNt/B than with BOTOX.RTM.,
although BoNt/B was less effective at weakening muscles. After four
months of injections 2 of 4 (where treated with 1.5 ng/kg) and 4 of
4 (where treated with 6.5 ng/kg) rabbits developed antibodies
against BoNt/B. In a separate study, 0 of 9 BOTOX.RTM. treated
rabbits demonstrated antibodies against BoNt/A. DAS results
indicate relative peak potencies of BoNt/A being equal to BoNt/F,
and BoNt/F being greater than BoNt/B. With regard to duration of
effect, BoNt/A was greater than BoNt/B, and BoNt/B duration of
effect was greater than BoNt/F. As shown by the therapeutic index
values, the two commercial preparations of BoNt/A (BOTOX.RTM. and
Dysport.RTM.) are different. The increased water consumption
behavior observed following hind limb injection of BoNt/B indicates
that clinically significant amounts of this serotype entered the
murine systemic circulation. The results also indicate that in
order to achieve efficacy comparable to BoNt/A, it is necessary to
increase doses of the other serotypes examined. Increased dosage
can comprise safety. Furthermore, in rabbits, serotype B was more
antigenic than was BOTOX.RTM., possibly because of the higher
protein load injected to achieve an effective dose of BoNt/B.
[0026] The tetanus neurotoxin acts mainly in the central nervous
system, while botulinum neurotoxin acts at the neuromuscular
junction; both act by inhibiting acetylcholine release from the
axon of the affected neuron into the synapse, resulting in
paralysis. The effect of intoxication on the affected neuron is
long-lasting and until recently has been thought to be
irreversible. The tetanus neurotoxin is known to exist in one
immunologically distinct serotype.
[0027] Acetylcholine
[0028] Typically only a single type of small molecule
neurotransmitter is released by each type of neuron in the
mammalian nervous system. The neurotransmitter acetylcholine is
secreted by neurons in many areas of the brain, but specifically by
the large pyramidal cells of the motor cortex, by several different
neurons in the basal ganglia, by the motor neurons that innervate
the skeletal muscles, by the preganglionic neurons of the autonomic
nervous system (both sympathetic and parasympathetic), by the
postganglionic neurons of the parasympathetic nervous system, and
by some of the postganglionic neurons of the sympathetic nervous
system. Essentially, only the postganglionic sympathetic nerve
fibers to the sweat glands, the piloerector muscles and a few blood
vessels are cholinergic and most of the postganglionic neurons of
the sympathetic nervous system secret the neurotransmitter
norepinephine. In most instances acetylcholine has an excitatory
effect. However, acetylcholine is known to have inhibitory effects
at some of the peripheral parasympathetic nerve endings, such as
inhibition of the heart by the vagal nerve.
[0029] The efferent signals of the autonomic nervous system are
transmitted to the body through either the sympathetic nervous
system or the parasympathetic nervous system. The preganglionic
neurons of the sympathetic nervous system extend from preganglionic
sympathetic neuron cell bodies located in the intermediolateral
horn of the spinal cord. The preganglionic sympathetic nerve
fibers, extending from the cell body, synapse with postganglionic
neurons located in either a paravertebral sympathetic ganglion or
in a prevertebral ganglion. Since, the preganglionic neurons of
both the sympathetic and parasympathetic nervous system are
cholinergic, application of acetylcholine to the ganglia will
excite both sympathetic and parasympathetic postganglionic
neurons.
[0030] Acetylcholine activates two types of receptors, muscarinic
and nicotinic receptors. The muscarinic receptors are found in all
effector cells stimulated by the postganglionic neurons of the
parasympathetic nervous system, as well as in those stimulated by
the postganglionic cholinergic neurons of the sympathetic nervous
system. The nicotinic receptors are found in the synapses between
the preganglionic and postganglionic neurons of both the
sympathetic and parasympathetic. The nicotinic receptors are also
present in many membranes of skeletal muscle fibers at the
neuromuscular junction.
[0031] Acetylcholine is released from cholinergic neurons when
small, clear, intracellular vesicles fuse with the presynaptic
neuronal cell membrane. A wide variety of non-neuronal secretory
cells, such as, adrenal medulla (as well as the PC12 cell line) and
pancreatic islet cells release catecholamines and insulin,
respectively, from large dense-core vesicles. The PC12 cell line is
a clone of rat pheochromocytoma cells extensively used as a tissue
culture model for studies of sympathoadrenal development. Botulinum
toxin inhibits the release of both types of compounds from both
types of cells in vitro, permeabilized (as by electroporation) or
by direct injection of the toxin into the denervated cell.
Botulinum toxin is also known to block release of the
neurotransmitter glutamate from cortical synaptosomes cell
cultures.
[0032] Sanders et al. in U.S. Pat. No. 5,766,605 disclose that
BoNT/A can be used to treat autonomic nervous system disorders, for
example rhinorrhea, otitis media, excessive salivation, asthma,
chronic obstructive pulmonary disease (COPD), excessive stomach
acid secretion, spastic colitis and excessive sweating.
[0033] Furthermore, Binder U.S. Pat. No. 5,714,468 discloses that
BoNT/A can be used to treat migraine headache pain that is
associated with muscle spasm, vascular disturbances, neuralgia and
neuropathy. Additionally, our laboratory data obtained from
experiments with rats show that pain, particularly inflammation
pain, may be reduced with an injection of botulinum serotype A,
either spinally or peripherally.
[0034] One of the reasons that BoNT/A has been selected over the
other serotypes, for example serotypes B, C.sub.1, D, E, F, and G,
for clinical use is that BoNT/A has a substantially longer lasting
therapeutic effect. In other words, the inhibitory effect of BoNT/A
is more persistent. Therefore, the other serotypes of botulinum
toxins could potentially be effectively used in a clinical
environment if their biological persistence could be enhanced. For
example, parotid sialocele is a condition where the patient suffers
from excessive salivation. Sanders et al. disclose in their patent
that serotype D may be very effective in reducing excessive
salivation. However, the biological persistence of serotype D
botulinum toxin is relatively short and thus may not be practical
for clinical use. If the biological persistence of serotype D may
be enhanced, it may effectively be used in a clinical environment
to treat, for example, parotid sialocele.
[0035] Another reason that BoNT/A has been a preferred neurotoxin
for clinical use is, as discussed above, its superb ability to
immobilize muscles through flaccid paralysis. For example, BoNT/A
is preferentially used to immobilize muscles and prevent limb
movements after a tendon surgery to facilitate recovery. However,
for some minor tendon surgeries, the healing time is relatively
short. It would be beneficial to have a BoNT/A without the
prolonged persistence for use in such circumstances so that the
patient can regain mobility at about the same time they recover
from the surgery.
[0036] There is a need to have modified neurotoxins that are non
serotype A botulinum toxins with enhanced biological persistence
and modified neurotoxins that are BoNT/A with reduced biological
persistence and methods for preparing such toxins.
SUMMARY
[0037] The present invention meets this need and provides for non
serotype A botulinum toxins with enhanced persistence and BoNT/A
with reduced persistence and methods for preparing such toxins.
[0038] In one broad embodiment of the invention, a modified
neurotoxin is formed from a neurotoxin which includes a structural
modification. The structural modification is able to alter the
biological persistence of the neurotoxin. In one embodiment, the
structural modification includes fusing a biological persistence
enhancing component with a neurotoxin. The biological persistence
enhancing component increases the duration of the inhibitory effect
of the modified neurotoxin intracellularly. Preferably, the
biological persistence enhancing component is a leucine-based
motif.
[0039] Without wishing to be limited by any particular theory or
mechanism of operation, it is believed that the leucine-based motif
enhances the persistence of a neurotoxin by increasing its
biological half-life. For example, it is known that BoNT/A has a
very long biological persistence. Keller et al., FEBS Letters,
456:137-142 (1999), investigated to determine whether the
persistence of BoNT/A is due to a depressed synthesis of SNAP-25 to
replace the cleaved ones, or is due to the stability of the light
chain intracellularly. Keller et al. found that the major factor
limiting cellular recovery is the prolonged stability of toxin, or
prolonged half-life.
[0040] Furthermore, without wishing to be limited by any particular
theory or mechanism of operation, it is believed that the
leucine-based motif located on the light chain, or the third amino
acid sequence region, of BoNT/A, and not on any other serotypes, is
responsible for the prolonged half-life of BoNT/A.
[0041] A leucine-based motif is often found on the carboxyl termini
of several membrane receptors and vesicular neurotransmitter
transporter and it apparently plays a crucial role in
vesicle/membrane trafficking. Liu et al. Trends Cell Biol,
9:356-363 (1999); Tan et al. J Biol Chem, 273:17351-17360 (1998);
Dietrich et al. J. Cell Bio, 138:271-281 (1997); Shin et al. J Biol
Chem, 266:10658-10665 (1991) and Geisler et al. J Biol Chem,
273:21316-21323 (1998). More specifically, the leuine-based motif
is found in a membrane-proximal, cytoplasmic, carboxylic terminal
tail of a membrane-bound receptor or transporter protein. It has
been demonstrated that adaptor proteins that are highly
concentrated at clathrin coated pits bind to the leucine-based
motif and that disruption of this motif disrupts endocytosis of the
motif-containing protein. Tan et al. J Biol Chem, 273:17351-17360
(1998); Dietrich et al. J. Cell Bio, 138:271-281 (1997) and Shin et
al. J Biol Chem, 266:10658-10665 (1991). Furthermore, addition of
the leucine-based motif to the carboxyl terminus of the plasma
membrane protein Tac resulted in endocytosis of the chimera,
suggesting that the motif is sufficient for targeted endocytosis.
Tan et al. Supra.
[0042] The leucine-based motif located on the light chain of BoNT/A
may cause the light chain to localize at the membranes, similarly
to how membrane-bound receptor or transporter protein are localized
at the membranes described above. Localization of the light chain
to the membrane may protect and preserve the light chain, and the
heavy chain if it is still attached, from being removed and/or
degraded by the intracellular cleaning processes, thereby rendering
it a long biological half-life. For example, intracellular
autophagosomes are responsible for cleaning the cytoplasm by
engulfing, and thereafter degrading, free floating foreign
substances in the cytoplasm. Erdal et al. Naunyn Schmiedebergs
Acrch Pharmacol, 351:67-78 (1995). Since the leucine-based motif
provides an anchor for the light chain, and the heavy chain if it
is still attached, it would be difficult for the autophagosomes to
remove and engulf the light chain from the cytoplasm. Thus the
light chain remains in the cytoplasm to continue exerting its
inhibitory effects on vesicular exocytosis of
neurotransmitters.
[0043] In another embodiment, the leucine-based motif located on
the light chain of BoNT/A is removed in its entirety or in parts.
This modified neurotoxin effectively has a shortened the biological
persistence. Preferably, this modified neurotoxin has a decreased
half-life.
[0044] This invention also provide for methods of producing
modified neurotoxins. Additionally, this invention provide for
methods of using the modified neurotoxins to treat biological
disorders.
DEFINITIONS
[0045] Before proceeding to describe the present invention, the
following definitions are provided and apply herein.
[0046] "Heavy chain" means the heavy chain of a clostridial
neurotoxin. It preferably has a molecular weight of about 100 kDa
and may be referred to herein as H chain or as H.
[0047] "H.sub.N" means a fragment (preferably having a molecular
weight of about 50 kDa) derived from the H chain of a Clostridial
neurotoxin which is approximately equivalent to the amino terminal
segment of the H chain, or the portion corresponding to that
fragment in the intact in the H chain. It is believed to contain
the portion of the natural or wild type clostridial neurotoxin
involved in the translocation of the L chain across an
intracellular endosomal membrane.
[0048] "H.sub.C" means a fragment (about 50 kDa) derived from the H
chain of a clostridial neurotoxin which is approximately equivalent
to the carboxyl terminal segment of the H chain, or the portion
corresponding to that fragment in the intact H chain. It is
believed to be immunogenic and to contain the portion of the
natural or wild type Clostridial neurotoxin involved in high
affinity, presynaptic binding to motor neurons.
[0049] "Light chain" means the light chain of a clostridial
neurotoxin. It preferably has a molecular weight of about 50 kDa,
and can be referred to as L chain, L or as the proteolytic domain
(amino acid sequence) of a clostridial neurotoxin. The light chain
is believed to be effective as an inhibitor of neurotransmitter
release when it is released into a cytoplasm of a target cell.
[0050] "Neurotoxin" means a molecule that is capable of interfering
with the functions of a neuron. The "neurotoxin" may be naturally
occurring or man-made.
[0051] "Modified neurotoxin" means a neurotoxin which includes a
structural modification. In other words, a "modified neurotoxin" is
a neurotoxin which has been modified by a structural modification.
The structural modification changes the biological persistence,
preferably the biological half-life, of the modified neurotoxin
relative to the neurotoxin from which the modified neurotoxin is
made. The modified neurotoxin is structurally different from a
naturally existing neurotoxin.
[0052] "Structural modification" means a physical change to the
neurotoxin that may be affected by covalently fusing one or more
amino acids to the neurotoxin. "Structural modification" also means
the deletion of one or more amino acids from a neurotoxin.
Furthermore, "structural modification" may also mean any changes to
a neurotoxin that makes it physically or chemically different from
an identical neurotoxin without the structural modification.
[0053] "Biological persistence" means the time duration in which a
neurotoxin or a modified neurotoxin causes an interference with a
neuronal function, for example the time duration in which a
neurotoxin or a modified neurotoxin causes a substantial inhibition
of the release of acetylcholine from a nerve terminal.
[0054] "Biological half-life" means the time that the concentration
of a neurotoxin or a modified neurotoxin, preferably the active
portion of the neurotoxin or modified neurotoxin, for example the
light chain of botulinum toxins, is reduced to half of the original
concentration in a mammal, preferably in the neurons of the
mammal.
DETAILED DESCRIPTION
[0055] The present invention is based upon the discovery that the
biological persistence of a neurotoxin may be altered by
structurally modifying the neurotoxin. In other words, a modified
neurotoxin with an altered biological persistence may be formed
from a neurotoxin containing or including a structural
modification. In one embodiment, the structural modification
includes the fusing a biological persistence enhancing component to
the primary structure of a neurotoxin to enhance its biological
persistence. In a preferred embodiment, the biological persistence
enhancing component is a leucine-based motif. Preferably, the
biological persistence enhancing component enhances the biological
half-life of the modified neurotoxin. More preferably, the
biological half-life of the modified neurotoxin is enhanced by
about 10%. Even more preferably, the biological half-life of the
modified neurotoxin is enhanced by about 100%. Generally speaking,
the modified neurotoxin has a biological persistence of about 20%
to 300% more than an identical neurotoxin without the structural
modification. That is, for example, the modified neurotoxin
including the biological persistence enhancing component is able to
cause a substantial inhibition of acetylcholine release from a
nerve terminal for about 20% to about 300% longer than a neurotoxin
that is not modified.
[0056] In a broad embodiment of the present invention, a
leucine-based motif is an oligomer of seven amino acids. The
oligomer is organized in to two groups. The first five amino acids
starting from the amino terminal of the leucine-based motif form a
"quintet of amino acids." The two amino acids immediately following
the quintet of amino acids form a "duplet of amino acids." In a
preferred embodiment, the duplet of amino acids is located at the
carboxyl terminal region of the leucine-based motif. In another
preferred embodiment, the quintet of amino acids includes at least
one acidic amino acid selected from a group consisting of a
glutamate and an aspartate.
[0057] The duplet of amino acid includes at least one hydrophobic
amino acid, for example leucine, isoleucine, methionine, alanine,
phenylalanine, tryptophan, valine or tyrosine. Preferably, the
duplet of amino acid is a leucine-leucine, a leucine-isoleucine, an
isoleucine-leucine or an isoleucine-isoleucine. Even more
preferably, the duplet is a leucine-leucine.
[0058] In one embodiment, the leucine-based motif is XDXXXLL (SEQ
ID NO: 1), wherein x and may be any amino acids. In another
embodiment, the leucine-based motif is XEXXXLL (SEQ ID NO: 2),
wherein E is glutamic acid. In another embodiment, the duplet of
amino acids may include an isoleucine or a methionine, forming
XDXXXLI (SEQ ID NO: 3) or XDXXXLM (SEQ ID NO: 4), respectively.
Additionally, the aspartic acid, D, may be replaced by a glutamic
acid, E, to form XEXXXLI (SEQ ID NO: 5) and XEXXXLM (SEQ ID NO: 6).
In a preferred embodiment, the leucine-based motif is SEQ ID NO:
7.
[0059] In another embodiment, the quintet of amino acids comprises
at least one hydroxyl containing amino acid, for example a serine,
a threonine or a tyrosine. Preferably, the hydroxyl containing
amino acid can be phosphorylated. More preferably, the hydroxyl
containing amino acid is a serine which can be phosphorylated to
allow for the binding of adaptor proteins.
[0060] Although non-modified amino acids are provided as examples,
a modified amino acid is also contemplated to be within the scope
of this invention. For example, leucine-based motif may include a
halogenated, preferably, fluorinated leucine.
[0061] Various leucine-based motif are found in various species. A
list of possible leucine-based motif derived from the various
species that may be used in accordance with this invention is shown
in Table 1. This list is not intended to be limiting.
TABLE-US-00001 TABLE 1 Species Sequence SEQ ID NO: BoNT/A FEFYKLL 7
Rat VMAT1 EEKRAIL 8 Rat VMAT 2 EEKMAIL 9 Rat VAChT SERDVLL 10 Rat
.delta. VDTQVLL 11 Mouse .delta. AEVQALL 12 Frog .gamma./.delta.
SDKQNLL 13 Chicken .gamma./.delta. SDRQNLI 14 Sheep .delta. ADTQVLM
15 Human CD3.gamma. SDKQTLL 16 Human CD4 SQIKRLL 17 Human .delta.
ADTQALL 18 VMAT is vesicular monoamine transporter; VACht is
vesicular acetylcholine transporter. Italicized serine residues are
potential sites of phosphorylation.
[0062] The modified neurotoxin may be formed from any neurotoxin.
Preferably, the neurotoxin used is a Clostridial neurotoxin. A
Clostridial neurotoxin comprises a polypeptide having three amino
acid sequence regions. The first amino acid sequence region
includes a neuronal binding moiety which is substantially
completely derived from a neurotoxin selected from a group
consisting of beratti toxin; butyricum toxin; tetani toxin; BoNT/A,
B, C.sub.1, D, E, F, and G. Preferably, the first amino acid
sequence region is derived from the carboxyl terminal region of a
toxin heavy chain, H.sub.C.
[0063] The second amino acid sequence region is effective to
translocate the polypeptide or a part thereof across an endosome
membrane into the cytoplasm of a neuron. In one embodiment, the
second amino acid sequence region of the polypeptide comprises an
amine terminal of a heavy chain, H.sub.N, derived from a neurotoxin
selected from a group consisting of beratti toxin; butyricum toxin;
tetani toxin; BoNT/A, B, C.sub.1, D, E, F, and G.
[0064] The third amino acid sequence region has therapeutic
activity when it is released into the cytoplasm of a target cell or
neuron. In one embodiment, the third amino acid sequence region of
the polypeptide comprises a toxin light chain, L, derived from a
neurotoxin selected from a group consisting of beratti toxin;
butyricum toxin; tetani toxin; BoNT/A, B, C.sub.1, D, E, F, and
G.
[0065] The Clostridial neurotoxin may be a hybrid neurotoxin. For
example, each of the neurotoxin's amino acid sequence regions may
be derived from a different Clostridial neurotoxin serotype. For
example, in one embodiment, the polypeptide comprises a first amino
acid sequence region derived from the H.sub.C of the tetani toxin,
a second amino acid sequence region derived from the H.sub.N of
BoNt/B, and a third amino acid sequence region derived from the L
chain of botulinum serotype E. All other possible combinations are
included within the scope of the present invention.
[0066] Alternatively, all three of the amino acid sequence regions
of the Clostridial neurotoxin may be from the same species and same
serotype. If all three amino acid sequence regions of the
neurotoxin are from the same Clostridial neurotoxin species and
serotype, the neurotoxin will be referred to by the species and
serotype name. For example, a neurotoxin polypeptide may have its
first, second and third amino acid sequence regions derived from
BoNT/E. In which case, the neurotoxin is referred as BoNT/E.
[0067] Additionally, each of the three amino acid sequence regions
may be modified from the naturally occurring sequence from which
they are derived. For example, the amino acid sequence region may
have at least one or more amino acid may be added or deleted as
compared to the naturally occurring sequence.
[0068] The biological persistence enhancing component, preferably
the leucine-based motif, may be fused with any of the above
described neurotoxin to form a modified neurotoxin with an enhanced
biological persistence. "Fusing" as used in the context of this
invention includes covalently adding to or covalently inserting in
between a primary structure of a neurotoxin. Preferably, the
biological persistence enhancing component is added to a
Clostridial neurotoxin which does not have a leucine-based motif in
its primary structure. For example, in one embodiment, the
leucine-based motif is fused with a hybrid neurotoxin, wherein the
third amino acid sequence is not derived from botulinum serotype A.
In another embodiment, the leucine-based motif is fused with a
BoNt/E.
[0069] In one embodiment, the leucine-based motif is fused with the
third amino acid sequence region of the neurotoxin. In a preferred
embodiment, the leucine-based motif is fused with the region
towards the carboxylic terminal of the third amino acid sequence
region. More preferably, the leucine-based motif is fused with the
carboxylic terminal of the third region of a neurotoxin. Even more
preferably, the leucine-based motif is fused with the carboxylic
terminal of the third region of BoNt/E.
[0070] In another embodiment, the structural modification of a
neurotoxin which has a preexisting leucine-based motif includes
deleting one or more amino acids from the leucine-based motif.
Alternatively, a modified neurotoxin includes a structural
modification which results in a neurotoxin with one or more amino
acids absent from the leucine-based motif. The removal of one or
more amino acids from the preexisting leucine-based motif is
effective to reduce the biological persistence of a modified
neurotoxin. More preferably, the deletion of one or more amino
acids from the leucine-based motif of BoNT/A reduces the biological
half-life of the modified neurotoxin.
[0071] In one broad aspect of the present invention, a method is
provided for treating a biological disorder using a modified
neurotoxin. The treatments may include treating neuromuscular
disorders, autonomic nervous system disorders and pain.
[0072] The neuromuscular disorders and conditions that may be
treated with a modified neurotoxin include: for example,
strabismus, blepharospasm, spasmodic torticollis (cervical
dystonia), oromandibular dystonia and spasmodic dysphonia
(largyngeal dystonia).
[0073] For example, Borodic U.S. Pat. No. 5,053,005 discloses
methods for treating juvenile spinal curvature, i.e. scoliosis,
using BoNT/A. The disclosure of Borodic is incorporated in its
entirety herein by reference. In one embodiment, using
substantially similar methods as disclosed by Borodic, a modified
neurotoxin is administered to a mammal, preferably a human, to
treat spinal curvature. In a preferred embodiment, a modified
neurotoxin comprising BoNT/E fused with a leucine-based motif is
administered. Even more preferably, a modified neurotoxin
comprising BoNT/E with a leucine-based motif fused to the carboxyl
terminal of its light chain is administered to the mammal,
preferably a human, to treat spinal curvature. The modified
neurotoxin may be administered to treat other neuromuscular
disorders using well known techniques that are commonly performed
with BoNT/A.
[0074] Autonomic nervous system disorders may also be treated with
a modified neurotoxin. For example, glandular malfunctioning is an
autonomic nervous system disorder. Glandular malfunctioning
includes excessive sweating and excessive salivation. Respiratory
malfunctioning is another example of an autonomic nervous system
disorder. Respiratory malfunctioning includes chronic obstructive
pulmonary disease and asthma. Sanders et al. discloses methods for
treating the autonomic nervous system, such as excessive sweating,
excessive salivation, asthma, etc., using naturally existing
botulinum toxins. The disclosure of Sander et al. is incorporated
in its entirety by reference herein. In one embodiment,
substantially similar methods to that of Sanders et al. may be
employed, but using a modified neurotoxin, to treat autonomic
nervous system disorders such as the ones discussed above. For
example, a modified neurotoxin may be locally applied to the nasal
cavity of the mammal in an amount sufficient to degenerate
cholinergic neurons of the autonomic nervous system that control
the mucous secretion in the nasal cavity.
[0075] Pain that may be treated by a modified neurotoxin include
pain caused by muscle tension, or spasm, or pain that is not
associated with muscle spasm. For example, Binder in U.S. Pat. No.
5,714,468 discloses that headache caused by vascular disturbances,
muscular tension, neuralgia and neuropathy may be treated with a
naturally occurring botulinum toxin, for example BoNT/A. The
disclosures of Binder is incorporated in its entirety herein by
reference. In one embodiment, substantially similar methods to that
of Binder may be employed, but using a modified neurotoxin, to
treat headache, especially the ones caused by vascular
disturbances, muscular tension, neuralgia and neuropathy. Pain
caused by muscle spasm may also be treated by an administration of
a modified neurotoxin. For example, a BoNT/E fused with a
leucine-based motif, preferably at the carboxyl terminal of the
BoNT/E light chain, may be administered intramuscularly at the
pain/spasm location to alleviate pain.
[0076] Furthermore, a modified neurotoxin may be administered to a
mammal to treat pain that is not associated with a muscular
disorder, such as spasm. In one broad embodiment, methods of the
present invention to treat non-spasm related pain include central
administration or peripheral administration of the modified
neurotoxin.
[0077] For example, Foster et al. in U.S. Pat. No. 5,989,545
discloses that a botulinum toxin conjugated with a targeting moiety
may be administered centrally (intrathecally) to alleviate pain.
The disclosures of Foster et al. is incorporated in its entirety by
reference herein. In one embodiment, substantially similar methods
to that of Foster et al. may be employed, but using the modified
neurotoxin according to this invention, to treat pain. The pain to
be treated may be an acute pain, or preferably, chronic pain.
[0078] An acute or chronic pain that is not associated with a
muscle spasm may also be alleviated with a local, peripheral
administration of the modified neurotoxin to an actual or a
perceived pain location on the mammal. In one embodiment, the
modified neurotoxin is administered subcutaneously at or near the
location of pain, for example at or near a cut. In another
embodiment, the modified neurotoxin is administered intramuscularly
at or near the location of pain, for example at or near a bruise
location on the mammal. In another embodiment, the modified
neurotoxin is injected directly into a joint of a mammal, for
treating or alleviating pain cause arthritis conditions. Also,
frequent repeated injections or infusion of the modified neurotoxin
to a peripheral pain location is within the scope of the present
invention. However, given the long lasting therapeutic effects of
the present invention, frequent injections or infusion of the
neurotoxin may not be necessary. For example, practice of the
present invention can provide an analgesic effect, per injection,
for 2 months or longer, for example 27 months, in humans.
[0079] Without wishing to limit the invention to any mechanism or
theory of operation, it is believed that when the modified
neurotoxin is administered locally to a peripheral location, it
inhibits the release of neuro-substances, for example substance P,
from the peripheral primary sensory terminal. Since the release of
substance P by the peripheral primary sensory terminal may cause or
at least amplify pain transmission process, inhibition of its
release at the peripheral primary sensory terminal will dampen the
transmission of pain signals from reaching the brain.
[0080] In addition to having pharmacologic actions at the
peripheral location, the modified neurotoxin of the present
invention may also have inhibitory effects in the central nervous
system. Presumably the retrograde transport is via the primary
afferent. This hypothesis is supported by our experimental data
which shows that BoNT/A is retrograde transported to the dorsal
horn when the neurotoxin is injected peripherally. Moreover, work
by Weigand et al, Nauny-Schmiedeberg's Arch. Pharmacol. 1976; 292,
161-165, and Habermann, Nauny-Schmiedeberg's Arch. Pharmacol. 1974;
281, 47-56, showed that botulinum toxin is able to ascend to the
spinal area by retrograde transport. As such, a modified
neurotoxin, for example BoNt/A with one or more amino acids deleted
from the leucine-based motif, injected at a peripheral location,
for example intramuscularly, may be retrograde transported from the
peripheral primary sensory terminal to the central primary sensory
terminal.
[0081] The amount of the modified neurotoxin administered can vary
widely according to the particular disorder being treated, its
severity and other various patient variables including size,
weight, age, and responsiveness to therapy. Generally, the dose of
modified neurotoxin to be administered will vary with the age,
presenting condition and weight of the mammal, preferably a human,
to be treated. The potency of the modified neurotoxin will also be
considered.
[0082] Assuming a potency which is substantially equivalent to
LD.sub.50=2,730 U in a human patient and an average person is 75
kg, a lethal dose would be about 36 U/kg of a modified neurotoxin.
Therefore, when a modified neurotoxin with such an LD.sub.50 is
administered, it would be appropriate to administer less than 36
U/kg of the modified neurotoxin into human subjects. Preferably,
about 0.01 U/kg to 30 U/kg of the modified neurotoxin is
administered. More preferably, about 1 U/kg to about 15 U/kg of the
modified neurotoxin is administered. Even more preferably, about 5
U/kg to about 10 U/kg modified neurotoxin is administered.
Generally, the modified neurotoxin will be administered as a
composition at a dosage that is proportionally equivalent to about
2.5 cc/100 U. Those of ordinary skill in the art will know, or can
readily ascertain, how to adjust these dosages for neurotoxin of
greater or lesser potency.
[0083] Although examples of routes of administration and dosages
are provided, the appropriate route of administration and dosage
are generally determined on a case by case basis by the attending
physician. Such determinations are routine to one of ordinary skill
in the art (see for example, Harrison's Principles of Internal
Medicine (1998), edited by Anthony Fauci et al., 14.sup.th edition,
published by McGraw Hill). For example, the route and dosage for
administration of a modified neurotoxin according to the present
disclosed invention can be selected based upon criteria such as the
solubility characteristics of the modified neurotoxin chosen as
well as the types of disorder being treated.
[0084] The modified neurotoxin may be produced by chemically
linking the leucine-based motif to a neurotoxin using conventional
chemical methods well known in the art. The neurotoxin may be
obtained from a harvesting neurotoxins. For example, BoNt/E can be
obtained by establishing and growing cultures of Clostridium
botulinum in a fermenter and then harvesting and purifying the
fermented mixture in accordance with known procedures. All the
botulinum toxin serotypes are initially synthesized as inactive
single chain proteins which must be cleaved or nicked by proteases
to become neuroactive. The bacterial strains that make botulinum
toxin serotypes A and G possess endogenous proteases and serotypes
A and G can therefore be recovered from bacterial cultures in
predominantly their active form. In contrast, botulinum toxin
serotypes C.sub.1, D and E are synthesized by nonproteolytic
strains and are therefore typically unactivated when recovered from
culture. Serotypes B and F are produced by both proteolytic and
nonproteolytic strains and therefore can be recovered in either the
active or inactive form. However, even the proteolytic strains that
produce, for example, the BoNt/B serotype only cleave a portion of
the toxin produced. The exact proportion of nicked to unnicked
molecules depends on the length of incubation and the temperature
of the culture. Therefore, a certain percentage of any preparation
of, for example, the BoNt/B toxin is likely to be inactive,
possibly accounting for the known significantly lower potency of
BoNt/B as compared to BoNt/A. The presence of inactive botulinum
toxin molecules in a clinical preparation will contribute to the
overall protein load of the preparation, which has been linked to
increased antigenicity, without contributing to its clinical
efficacy. Additionally, it is known that BoNt/B has, upon
intramuscular injection, a shorter duration of activity and is also
less potent than BoNt/A at the same dose level.
[0085] The modified neurotoxin may also be produced by recombinant
techniques. Recombinant techniques are preferable for producing a
neurotoxin having amino acid sequence regions from different
Clostridial species or having modified amino acid sequence regions.
Also, the recombinant technique is preferable in producing BoNT/A
with the leucine-based motif being modified by deletion. The
technique includes steps of obtaining genetic materials from
natural sources, or synthetic sources, which have codes for a
neuronal binding moiety, an amino acid sequence effective to
translocate the neurotoxin or a part thereof, and an amino acid
sequence having therapeutic activity when released into a cytoplasm
of a target cell, preferably a neuron. In a preferred embodiment,
the genetic materials have codes for the biological persistence
enhancing component, preferably the leucine-based motif, the
H.sub.C, the H.sub.N and the L chain of the Clostridial neurotoxins
and fragments thereof. The genetic constructs are incorporated into
host cells for amplification by first fusing the genetic constructs
with a cloning vectors, such as phages or plasmids. Then the
cloning vectors are inserted into hosts, preferably E. coli's.
Following the expressions of the recombinant genes in host cells,
the resultant proteins can be isolated using conventional
techniques.
[0086] There are many advantages to producing these modified
neurotoxins recombinantly. For example, to form a modified
neurotoxin, a modifying fragment must be attached or inserted into
a neurotoxin. The production of neurotoxin from anaerobic
Clostridium cultures is a cumbersome and time-consuming process
including a multi-step purification protocol involving several
protein precipitation steps and either prolonged and repeated
crystallization of the toxin or several stages of column
chromatography. Significantly, the high toxicity of the product
dictates that the procedure must be performed under strict
containment (BL-3). During the fermentation process, the folded
single-chain neurotoxins are activated by endogenous clostridial
proteases through a process termed nicking to create a dichain.
Sometimes, the process of nicking involves the removal of
approximately 10 amino acid residues from the single-chain to
create the dichain form in which the two chains remain covalently
linked through the intrachain disulfide bond.
[0087] The nicked neurotoxin is much more active than the unnicked
form. The amount and precise location of nicking varies with the
serotypes of the bacteria producing the toxin. The differences in
single-chain neurotoxin activation and, hence, the yield of nicked
toxin, are due to variations in the serotype and amounts of
proteolytic activity produced by a given strain. For example,
greater than 99% of Clostridial botulinum serotype A single-chain
neurotoxin is activated by the Hall A Clostridial botulinum strain,
whereas serotype B and E strains produce toxins with lower amounts
of activation (0 to 75% depending upon the fermentation time).
Thus, the high toxicity of the mature neurotoxin plays a major part
in the commercial manufacture of neurotoxins as therapeutic
agents.
[0088] The degree of activation of engineered clostridial toxins
is, therefore, an important consideration for manufacture of these
materials. It would be a major advantage if neurotoxins such as
botulinum toxin and tetanus toxin could be expressed,
recombinantly, in high yield in rapidly-growing bacteria (such as
heterologous E. coli cells) as relatively non-toxic single-chains
(or single chains having reduced toxic activity) which are safe,
easy to isolate and simple to convert to the fully-active form.
[0089] With safety being a prime concern, previous work has
concentrated on the expression in E. coli and purification of
individual H and L chains of tetanus and botulinum toxins; these
isolated chains are, by themselves, non-toxic; see Li et al.,
Biochemistry 33:7014-7020 (1994); Zhou et al., Biochemistry
34:15175-15181 (1995), hereby incorporated by reference herein.
Following the separate production of these peptide chains and under
strictly controlled conditions the H and L chains can be combined
by oxidative disulphide linkage to form the neuroparalytic
di-chains.
EXAMPLES
[0090] The following non-limiting examples provide those of
ordinary skill in the art with specific preferred methods to treat
non-spasm related pain within the scope of the present invention
and are not intended to limit the scope of the invention.
Example 1
Treatment of Pain Associated with Muscle Disorder
[0091] An unfortunate 36 year old woman has a 15 year history of
temporomandibular joint disease and chronic pain along the masseter
and temporalis muscles. Fifteen years prior to evaluation she noted
increased immobility of the jaw associated with pain and jaw
opening and closing and tenderness along each side of her face. The
left side is originally thought to be worse than the right. She is
diagnosed as having temporomandibular joint (TMJ) dysfunction with
subluxation of the joint and is treated with surgical orthoplasty
meniscusectomy and condyle resection.
[0092] She continues to have difficulty with opening and closing
her jaw after the surgical procedures and for this reason, several
years later, a surgical procedure to replace prosthetic joints on
both sides is performed. After the surgical procedure progressive
spasms and deviation of the jaw ensues. Further surgical revision
is performed subsequent to the original operation to correct
prosthetic joint loosening. The jaw continues to exhibit
considerable pain and immobility after these surgical procedures.
The TMJ remained tender as well as the muscle itself. There are
tender points over the temporomandibular joint as well as increased
tone in the entire muscle. She is diagnosed as having post-surgical
myofascial pain syndrome and is injected with 7 U/kg of the
modified neurotoxin into the masseter and temporalis muscles,
preferably the modified neurotoxin is BoNT/E fused with a
leucine-based motif.
[0093] Several days after the injections she noted substantial
improvement in her pain and reports that her jaw feels looser. This
gradually improves over a 2 to 3 week period in which she notes
increased ability to open the jaw and diminishing pain. The patient
states that the pain is better than at any time in the last 4
years. The improved condition persists for up to 27 months after
the original injection of the modified neurotoxin.
Example 2
Treatment of Pain Subsequent to Spinal Cord Injury
[0094] A patient, age 39, experiencing pain subsequent to spinal
cord injury is treated by intrathecal administration, for example
by spinal tap or by catherization (for infusion), to the spinal
cord, with between about 0.1 U/kg of the modified neurotoxin,
preferably the modified neurotoxin is BoNT/E fused with a
leucine-based motif. The particular toxin dose and site of
injection, as well as the frequency of toxin administrations depend
upon a variety of factors within the skill of the treating
physician, as previously set forth. Within about 1 to about 7 days
after the modified neurotoxin administration, the patient's pain is
substantially reduced. The pain alleviation persists for up to 27
months.
Example 3
Peripheral Administration of a Modified Neurotoxin to Treat
"Shoulder-Hand Syndrome"
[0095] Pain in the shoulder, arm, and hand can develop, with
muscular dystrophy, osteoporosis, and fixation of joints. While
most common after coronary insufficiency, this syndrome may occur
with cervical osteoarthritis or localized shoulder disease, or
after any prolonged illness that requires the patient to remain in
bed.
[0096] A 46 year old woman presents a shoulder-hand syndrome type
pain. The pain is particularly localized at the deltoid region. The
patient is treated by a bolus injection of between about 0.05 U/kg
to about 2 U/kg of a modified neurotoxin subcutaneously to the
shoulder, preferably the modified neurotoxin is BoNT/E fused with a
leucine-based motif. The particular dose as well as the frequency
of administrations depends upon a variety of factors within the
skill of the treating physician, as previously set forth. Within
1-7 days after modified neurotoxin administration the patient's
pain is substantially alleviated. The duration of the pain
alleviation is from about 7 to about 27 months.
Example 4
Peripheral Administration of a Modified Neurotoxin to Treat
Postherpetic Neuralgia
[0097] Postherpetic neuralgia is one of the most intractable of
chronic pain problems. Patients suffering this excruciatingly
painful process often are elderly, have debilitating disease, and
are not suitable for major interventional procedures. The diagnosis
is readily made by the appearance of the healed lesions of herpes
and by the patient's history. The pain is intense and emotionally
distressing. Postherpetic neuralgia may occur any where, but is
most often in the thorax.
[0098] A 76 year old man presents a postherpetic type pain. The
pain is localized to the abdomen region. The patient is treated by
a bolus injection of between about 0.05 U/kg to about 2 U/kg of a
modified neurotoxin intradermally to the abdomen, preferably the
modified neurotoxin is BoNT/E fused with a leucine-based motif. The
particular dose as well as the frequency of administrations depends
upon a variety of factors within the skill of the treating
physician, as previously set forth. Within 1-7 days after modified
neurotoxin administration the patient's pain is substantially
alleviated. The duration of the pain alleviation is from about 7 to
about 27 months.
Example 5
Peripheral Administration of a Modified Neurotoxin to Treat
Nasopharyngeal Tumor Pain
[0099] These tumors, most often squamous cell carcinomas, are
usually in the fossa of Rosenmuller and may invade the base of the
skull. Pain in the face is common. It is constant, dull-aching in
nature.
[0100] A 35 year old man presents a nasopharyngeal tumor type pain.
Pain is found at the lower left cheek. The patient is treated by a
bolus injection of between about 0.05 U/kg to about 2 U/kg of a
modified neurotoxin intramuscularly to the cheek, preferably the
modified neurotoxin is BoNT/E fused with a leucine-based motif. The
particular dose as well as the frequency of administrations depends
upon a variety of factors within the skill of the treating
physician, as previously set forth. Within 1-7 days after modified
neurotoxin administration the patient's pain is substantially
alleviated. The duration of the pain alleviation is from about 7 to
about 27 months.
Example 6
Peripheral Administration of a Modified Neurotoxin to Treat
Inflammatory Pain
[0101] A patient, age 45, presents an inflammatory pain in the
chest region. The patient is treated by a bolus injection of
between about 0.05 U/kg to about 2 U/kg of a modified neurotoxin
intramuscularly to the chest, preferably the modified neurotoxin is
BoNT/E fused with a leucine-based motif. The particular dose as
well as the frequency of administrations depends upon a variety of
factors within the skill of the treating physician, as previously
set forth. Within 1-7 days after modified neurotoxin administration
the patient's pain is substantially alleviated. The duration of the
pain alleviation is from about 7 to about 27 months.
Example 7
Treatment of Excessive Sweating
[0102] A male, age 65, with excessive unilateral sweating is
treated by administering 0.05 U/kg to about 2 U/kg of a modified
neurotoxin, depending upon degree of desired effect. Preferably the
modified neurotoxin is BoNT/E fused with a leucine-based motif. The
administration is to the gland nerve plexus, ganglion, spinal cord
or central nervous system. The specific site of administration is
to be determined by the physician's knowledge of the anatomy and
physiology of the target glands and secretary cells. In addition,
the appropriate spinal cord level or brain area can be injected
with the toxin. The cessation of excessive sweating after the
modified neurotoxin treatment is up to 27 months.
Example 8
Post Surgical Treatments
[0103] A female, age 22, presents a torn shoulder tendon and
undergoes orthopedic surgery to repair the tendon. After the
surgery, the patient is administered intramuscularly with about
0.05 U/kg to about 2 U/kg of a modified neurotoxin to the shoulder.
Preferably, the modified neurotoxin is a BoNT/A wherein the
leucine-based motif is removed. The specific site of administration
is to be determined by the physician's knowledge of the anatomy and
physiology of the muscles. The administered modified neurotoxin
reduces movement of the arm to facilitate the recovery from the
surgery. The effect of the modified neurotoxin is for about five
weeks.
Example 9
Production of a Modified Neurotoxin with an Enhanced Biological
Persistence
[0104] A modified neurotoxin may be produced by employing
recombinant techniques in conjunction with conventional chemical
techniques.
[0105] The neurotoxin that is to be fused with the leucine-based
motif to form a modified neurotoxin may be produced recombinantly.
The recombinant technique includes steps of obtaining genetic
materials from either DNA cloned from natural sources, or synthetic
oligonucleotide sequences, which have codes for a neurotoxin,
preferably BoNT/E. The genetic constructs are incorporated into
host cells for amplification by first fusing the genetic constructs
with a cloning vectors, such as phages or plasmids. Then the
cloning vectors are inserted into hosts, preferably E. coli's.
Following the expressions of the recombinant genes in host cells,
the resultant proteins can be isolated using conventional
techniques.
[0106] The neurotoxin, preferably BoNT/E, derived from the
recombinant techniques can then be covalently fused with a
leucine-based motif. Preferably, the leucine-based motif is fused
to the light chain of BoNT/E at the carboxyl terminal. The fusion
of the leucine-based motif with BoNT/E is achieved via chemical
coupling using reagents and techniques known to those skilled in
the art, for example PDPH/EDAC and Traut's reagent chemistry.
[0107] The modified neurotoxin produced according to this example
has an enhanced biological persistence. Preferably, the biological
persistence is enhanced by about 20% to about 300% relative to an
identical neurotoxin without a leucine-based motif.
Example 10
Production of a Modified Neurotoxin with a Reduced Biological
Persistence
[0108] A modified neurotoxin with a reduced biological persistence
may be produced by employing recombinant techniques. The
recombinant technique includes steps of obtaining genetic materials
from a synthetic oligonucleotide sequences, which have codes for a
neurotoxin, preferably BoNT/A, which does not have genetic codings
for a leucine-based motif. The genetic constructs are incorporated
into host cells for amplification by first fusing the genetic
constructs with a cloning vectors, such as phages or plasmids. Then
the cloning vectors are inserted into hosts, preferably E. coli's.
Following the expressions of the recombinant genes in host cells,
the resultant proteins can be isolated using conventional
techniques.
[0109] The modified neurotoxin produced according to this example
has a reduced biological persistence. Preferably, the biological
persistence is reduced by about 20% to about 300% relative to an
identical neurotoxin, for example BoNT/A, with the leucine-based
motif.
[0110] Although the present invention has been described in detail
with regard to certain preferred methods, other embodiments,
versions, and modifications within the scope of the present
invention are possible. For example, a wide variety of modified
neurotoxins can be effectively used in the methods of the present
invention in place of clostridial neurotoxins. Also, the
corresponding genetic codes, i.e. DNA sequence, to the modified
neurotoxins are also considered to be part of this invention.
Additionally, the present invention includes peripheral
administration methods wherein two or more modified neurotoxins,
for example BoNT/E with a fused leucine-based motif and BoNT/B with
fused leucine-based motif, are administered concurrently or
consecutively. While this invention has been described with respect
to various specific examples and embodiments, it is to be
understood that the invention is not limited thereto and that it
can be variously practiced with the scope of the following claims.
Sequence CWU 1
1
1817PRTArtificial SequenceConsensus sequence for Leucine-based
motif. 1Xaa Asp Xaa Xaa Xaa Leu Leu1 527PRTArtificial
SequenceConsensus sequence for Leucine-based motif. 2Xaa Glu Xaa
Xaa Xaa Leu Leu1 537PRTArtificial SequenceConsensus sequence for
Leucine-based motif. 3Xaa Asp Xaa Xaa Xaa Leu Ile1 547PRTArtificial
SequenceConsensus sequence for Leucine-based motif. 4Xaa Asp Xaa
Xaa Xaa Leu Met1 557PRTArtificial SequenceConsensus sequence for
Leucine-based motif. 5Xaa Glu Xaa Xaa Xaa Leu Ile1 567PRTArtificial
SequenceConsensus sequence for Leucine-based motif. 6Xaa Glu Xaa
Xaa Xaa Leu Met1 577PRTClostridial botulinum serotype A 7Phe Glu
Phe Tyr Lys Leu Leu1 587PRTRattus norvegicus 8Glu Glu Lys Arg Ala
Ile Leu1 597PRTRattus norvegicus 9Glu Glu Lys Met Ala Ile Leu1
5107PRTRattus norvegicus 10Ser Glu Arg Asp Val Leu Leu1
5117PRTRattus norvegicus 11Val Asp Thr Gln Val Leu Leu1 5127PRTMus
musculus 12Ala Glu Val Gln Ala Leu Leu1 5137PRTXenopus laevis 13Ser
Asp Lys Gln Asn Leu Leu1 5147PRTGallus gallus 14Ser Asp Arg Gln Asn
Leu Ile1 5157PRTSheep 15Ala Asp Thr Gln Val Leu Met1 5167PRTHomo
sapiens 16Ser Asp Lys Asn Thr Leu Leu1 5177PRTHomo sapiens 17Ser
Gln Ile Lys Arg Leu Leu1 5187PRTHomo sapiens 18Ala Asp Thr Gln Ala
Leu Leu1 5
* * * * *